Human interferon alpha (IFN-α), also known as leukocyte interferon and a interferon, comprises a family of extracellular signaling proteins with antiviral, antiproliferative, and immunomodulatory activities. The first type of interferon to be identified and commercialized, IFN-α remains the most widely used interferon for clinical applications.
IFN-α is a member of the family of Type I interferons, which also includes IFN-β, omega (leukocyte (II)) interferon and tau (trophoblast) interferon. Omega and tau interferons are not clinically used. IFN-β, also known as fibroblast interferon, is well characterized but less utilized than IFN-α in the clinic. Fibroblasts are the predominant cellular producers of IFN-β. IFN-β has been approved in the United States for the treatment of relapsing forms of multiple sclerosis. Interferon gamma (IFN-γ), also known as gamma interferon, is the only known type II interferon. IFN-γ is produced by activated T lymphocytes and plays an important role in the establishment of a Th1 immune response. Its therapeutic use is limited. In the United States, human IFN-γ has been approved for reducing the frequency and severity of infections with chronic granulomatous disease.
IFN-α itself represents a family of more than a dozen related, homologous proteins (isoforms, Table 1), each encoded by a unique gene and each exhibiting a unique activity profile. The activities of the different α interferon species on viruses can vary as much as twenty-fold or more.
IFN-α products in clinical use are recombinant proteins or highly purified natural proteins of a single isoform. Recombinant IFN-α has been approved for use in the treatment of a variety of tumors and viral diseases (Table 2).
Until recently, B lymphocytes were believed to be the predominant producers of IFN-α. Recently a new cell type has been identified in the peripheral blood as the major source of Type I interferon production. These previously unidentified “natural interferon producing cells” (IPC) had been described for many years as a rare CD4+/MHC class II+ population (1:1000 within peripheral blood mononuclear cells (PBMC)) capable of synthesizing extremely large amounts of type I IFN upon viral infection. Cella M et al. Nat Med 5:919 (1999); Galy A et al. Blood 95:128 (2000); Siegal F P et al. Science 284:1835 (1999). After isolation of IPCs from the peripheral blood, IL-3 is required for survival of this cell type.
TABLE 1Family of Human IFN-αIFN-αA(IFN-α2a)IFN-α2(IFN-α2b)IFN-α4b(IFN-α4)IFN-αB2(IFN-α8)IFN-αC(IFN-α10)IFN-αD(IFN-α1)IFN-αF(IFN-α21)IFN-αG(IFN-α5)IFN-αH2(IFN-α14)IFN-αI(IFN-α17)IFN-αJ1(IFN-α7)IFN-αK(IFN-α6)IFN-αM1IFN-αNIFN-αWA(IFN-α16)
TABLE 2Current Clinical Approval of IFN-αApproved Outside theApproved in the United StatesUnited StatesChronic hepatitis BMultiple myelomaChronic hepatitis CRenal cell carcinomaHairy cell leukemiaBladder cell carcinomaCutaneous T-cell leukemiaColon carcinomaChronic myeloid leukemiaCervical dysplasiaNon-Hodgkin's lymphomaLaryngeal papillomatosisAdjuvant therapy for malignant melanomaKaposi's Sarcoma (AIDS-related)Condylomata acuminata (venereal warts)
Dendritic cells (DC) are thought to play a key role in the priming of immune responses against neoantigens. Banchereau J et al., Nature 392:245 (1998). Recent evidence suggests the presence of several distinct DC subtypes in human peripheral blood. Zhong R K et al. J Immunol 163:1254 (1999). These subtypes of DC include myeloid DC (mDC) and plasmacytoid DC (pDC, also known as DC2 cells). Precursor dendritic cells contain two subsets, a CD11c+/CD123+/− population (precursor of mDC) and a CD11c−/CD123++ population (precursor of pDC). The latter has recently attracted major attention since it was reported to be identical with the natural type I IFN producing cell (IPC). O'Doherty U et al. J Exp Med 178:1067 (1993); Grouard G et al. J Exp Med 185:1101 (1997); Thomas R et al. J Immunol 153:4016 (1994). Upon maturation this cell type develops characteristic features of DC. O'Doherty U et al. J Exp Med 178:1067 (1993); Thomas R et al. J Immunol 153:4016 (1994); Galy A et al. Blood 95:128 (2000); Chehimi J et al. Immunology 68:488. (1989).
The frequency of IPCs in PBMC in normal individuals varies between 0.2 and 0.6 percent. They are characterized by the absence of lineage markers CD3 (T cells), CD14 (monocytes), CD19 (B cells) and CD56 (NK cells), by the absence of CD11c, and by their expression of CD4, CD123 (IL-3 receptor α, IL-3Rα) and MHC class II. Grouard G et al. J Exp Med 185:1101-11 (1997); Rissoan M-C et al. Science 283:1183-6 (1999); Siegal F P et al. Science 284:1835-7 (1999); Cella M et al. Nat Med 5:919-23 (1999). Morphologically IPCs resemble lymphocytes. IPCs can be isolated from PBMC by a combination of magnetic bead activated cell sorting (MACS) and fluorescence-activated cell sorting (flow cytometry, FACS). Without addition of IL-3, most of the IPCs die within 3 days of cell culture. Infection of IPCs with herpes simplex virus (HSV, Siegal F P et al. Science 284:1835-7 (1999)) or influenza virus (Cella M et al. Nat Med 5:919-23 (1999)) leads to production of large amounts of type I interferons as measured by a bioassay (protection of fibroblasts against vesicular stomatitis virus).
Aside from its role in carrying the genetic code, DNA has recently been shown to function as a signaling molecule (Krieg A M, 1998, Biodrugs). The immune systems of higher eukaryotes appear to have evolved a mechanism to detect prokaryotic nucleic acids based on their content of unmethylated CpG dinucleotides in particular base contexts. Krieg A M et al. Nature 374:546-9 (1995). Unmethylated CpG dinucleotides are common in bacterial DNA, but are underrepresented (“CpG suppression”) and are methylated in vertebrate DNA. Bird A P Trends in Genetics 3:342 (1987). DNA containing these unmethylated CpG dinucleotides in immune stimulatory base contexts (“CpG motifs”) triggers humoral immunity by inducing B cell activation, resistance to activation-induced apoptosis, and IL-6 and IgM secretion. Krieg A M et al. Nature 374:546-9 (1995); Yi A K et al. J Immunol 157:5394 (1996); and Klinman D et al. Proc Natl Acad Sci USA 93:2879 (1996). Such CpG DNA also directly activates monocytes and macrophage to secrete Th1-like cytokines. Ballas Z K et al. J Immunol 157:1840 (1996); Cowdery J S et al. J Immunol 156:4570 (1996); and Halpern M D et al. Cell Immunol 167:72 (1996). This leads to the activation of natural killer (NK) cell lytic activity and IFN-γ secretion. Ballas Z K et al. J Immunol 157:1840 (1996); Cowdery J S et al. J Immunol 156:4570 (1996); and Chace J H Clin Immunol Immunopath 84:185-93 (1997).
Yamamoto et al. reported in 1988 their findings that a nucleic acid fraction, designated MY-1, extracted from Mycobacterium bovis (BCG) induced type I interferon in vitro. Yamamoto S et al. Jpn J Cancer Res 79:866-73 (1988). Subsequently Tokunaga et al. subsequently synthesized a panel of 45-mer oligonucleotides with sequence present in cDNA encoding three randomly selected known BCG proteins and found that one sequence, BCG-A4, was a strong inducer of type I IFN in mouse spleen cell suspensions. Tokunaga T et al. Microbiol Immunol 36:55-66 (1992). A 5′ 30-mer fragment, BCG-A4a, was reported to be as potent an inducer of IFN as the intact 45-mer BCG-A4.
BCG-A4ACCGATGACGTCGCCGGTGACGGCACCACGACGG(SEQ ID NO:163)CCACCGTGCTG BCG-A4aACCGATGACGTCGCCGGTGACGGCACCACG(SEQ ID NO:164)These workers went on to report that all oligonucleotides that induced IFN included a hexamer palindromic sequence GACGTC (present in BCG-A4 and BCG-A4a), AGCGCT, and AACGTT, but not ACCGGT. Yamamoto S et al. J Immunol 148:4072-6 (1992). Kimura et al. then found that among 30-mer phosophodiester oligodeoxynucleotides (ODNs) containing the hexamer palindrome AACGTT and oligoA, oligoC, oligoT, and oligoG ends, the latter (GGGGGGGGGGGGAACGTTGGGGGGGGGGGG; SEQ ID NO:165) was the strongest inducer of type I IFN in mouse spleen cell suspensions. Kimura Y et al. J Biochem (Tokyo) 116:991-4 (1994).
Recently it was surprisingly discovered that CpG ODN sequences with the strongest activity on human B cells did not induce detectable levels of type I IFN in PBMC. Hartmann G et al. J Immunol 164:1617-24 (2000).